Immunization of fish with plant-expressed recombinant proteins

ABSTRACT

Plants are produced that express an amino acid sequence that, when administered to a fish, produce an antigenic or immune response in the fish. The amino acid sequence in one embodiment is an antigen from an organism that causes pathology in fish. The plant tissue may be fed to the fish, or mixed with other materials and fed to fish, or extracted and administered to the fish.

[0001] This application claims priority to U.S. patent application Ser.No. 60/433,381 filed on Dec. 13, 2002 and is incorporated by referencein its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to the expression of fish disease antigensin transgenic plants and the use of the same as a vaccine.

BACKGROUND OF THE INVENTION

[0003] Over the past decade, transgenic plants have been successfullyused to express a variety of useful proteins. For example, production ofproteases in plants has been achieved (See U.S. Pat. No. 6,087,558);along with production of aprotinin in plants (U.S. Pat. No. 5,824,870);and avidin (U.S. Pat. No 5,767,379). A variety of mammalian bacterialand viral pathogen antigens are included in those proteins that havebeen successfully produced in plants, such as viral vaccines (U.S. Pat.No. 6,136,320), transmissible gastroenteritis and hepatitis vaccines(U.S. Pat. Nos. 5,914,123 and 6,034,298). These patents, as well as allreferences cited herein are incorporated herein by reference.

[0004] Many of the resulting peptides induced an immunogenic response inmice (Mason et al. (1998) Vaccine 16:1336-1343; Wigdorovitz et al.(1999) Virology 155:347-353), and humans (Kapusta et al. (1999) FASEB J.13:1796-1799) comparable to that of the original pathogen. After oraldelivery, these edible vaccines were immunogenic and could induceprotection. Mice fed a basic diet plus corn expressing recombinantEscherichia coli heat-labile enterotoxin B-subunit (LtB) mounted a gooddose dependent IgG and IgA response (Streatfield et al. “Plant basedvaccines—unique advances” Vaccine (2001)19:2742-2748.) Some of the firstedible vaccine technologies developed include transgenic potatoesexpressing hepatitis, TGEV and Norwalk virus antigens as well as variousother viral antigens. (See, e.g., Thanavala et al. (1995) Proc. Natl.Acad. Sci. U.S.A. 92:3358-3361; U.S. Pat. No. 6,136,320; U.S. Pat. No.6,034,298; U.S. Pat. No. 5,914,123; U.S. Pat. No. 5,612,487 and U.S.Pat. No. 5,484,719; Mason et al., (1996) Proc. Natl. Acad. Sci.93:5335-5340; “VP1 protein for foot-and-mouth disease” (Wigdorovitz etal (1999) Virology 255:347-353).

[0005] The utilization of transgenic plants for vaccine production hasseveral potential benefits over traditional vaccine production methods.First, transgenic plants are usually constructed to express only a smallantigenic portion of the pathogen or toxin, eliminating the possibilityof infection or innate toxicity of the whole organism and reducing thepotential for adverse reactions. Second, since there are no known humanor animal pathogens that are able to infect plants, concerns with viralor prion contamination are eliminated. Third, immunogen production intransgenic crops relies on the same established technologies to sow,harvest, store, transport, and process the plant as those commonly usedfor food crops, making transgenic plants a very economical means oflarge-scale vaccine production. Fourth, expression of immunogens in thenatural protein-storage compartments of plants maximizes stability,minimizes the need for refrigeration and keeps transportation andstorage costs low. Fifth, formulation of multicomponent vaccines ispossible by blending the seed of multiple transgenic plant lines into asingle vaccine. Sixth, direct oral administration is possible whenimmunogens are expressed in commonly consumed food plants, such asgrain, leading to the production of edible vaccines.

[0006] Oral vaccine delivery as the primary or booster immunization isby far the most sought after method by the aquaculture industry becauseit is suitable for the mass immunization of fish of all sizes, it isless stressful on fish than injection delivery, which requires handlingof the fish, and because it induces mucosal immunity. However thecost-effectiveness of oral delivery has been a major barrier tocommercialization of this method, especially for larger fish. Efficacyof oral antigen delivery is reported to be limited by the destructionand absorption of the antigens by the fish digestive system.

[0007] The inventors have found that transgenic plants can provide anideal system for economical production of antigens for oral vaccinationof fish.

BRIEF DESCRIPTION OF THE INVENTION

[0008] According to one aspect of the invention there is provided use ofa plant-derived recombinant amino acid sequence in the manufacture of amedicament for the prevention or treatment of disease in fish, whereinthe amino acid sequence, when administered to fish, produces anantigenic or immunogenic response in the fish. Preferably therecombinant amino acid sequence is an antigen of an organism that causesdisease or pathology in fish.

[0009] In one aspect of the invention a plant is transformed with anucleotide sequence encoding an amino acid sequence which, whenadministered to a fish, produces an antigenic or immunogenic response inthe fish.

[0010] In a further aspect of the invention, expression of the aminoacid sequence is preferentially directed to the seed of the plant.

[0011] In another aspect, the invention provides an amino acid sequencederived by expression in a plant cell, wherein said amino acid sequenceis endogenous to an organism causing disease or pathology in fish.

[0012] In another aspect, the invention provides a composition suitablefor oral delivery to fish, comprising a plant-derived recombinant aminoacid sequence, in particular a plant-derived recombinant amino acidsequence which is an antigen of an organism that causes disease orpathology in a fish.

[0013] In yet another aspect, the invention provides a method ofimmunizing fish against disease, which comprises administering to a fisha composition comprising a plant-derived recombinant amino acid sequencewhich is an antigen of an organism that causes disease or pathology in afish.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 shows the barley alpha amylase sequence fused to a sequenceencoding the avidin mature protein (SEQ ID NO: 1).

[0015]FIG. 2 is a plasmid map of pPHI5158.

[0016]FIG. 3 shows the maize optimized pat sequence (SEQ ID NO: 2).

[0017]FIG. 4 is a plasmid map of PGN7101.

[0018]FIG. 5A is the nucleotide sequence of maize codon optimized LtB(SEQ ID NO: 3).

[0019]FIG. 5B is the nucleotide sequence of BAASS:LtB (SEQ ID NO: 4).

[0020]FIG. 6 is the nucleotide sequence of IPNV VP2 (SEQ ID NO: 5).

[0021]FIG. 7 is the nucleotide sequence of BAASS:VP2 (SEQ ID NO: 6).

[0022]FIG. 8 is the nucleotide sequence of IPNV VP3 (SEQ ID NO: 7).

[0023]FIG. 9 is the nucleotide sequence of BAASS:VP3 (SEQ ID NO: 8).

[0024]FIG. 10 is the plasmid map of PGN9084.

[0025]FIG. 11 is the plasmid map of PGN9111.

[0026]FIG. 12 is a Western blot of the VP2 and VP3 proteins expressed inseed, resulting from event NVA.

[0027]FIG. 13 is a Western blot of the VP2 and VP3 proteins expressed inseed, resulting from event NVB.

[0028]FIG. 14 is a graph showing mean weight of fish at the time 0(first bar) and 8 weeks after vaccination (second bar). Standard errorbars are also shown

[0029]FIG. 15 are graphs showing mean antibody response of Atlanticsalmon at 8 weeks post-injection or feeding of recombinant avidin (A) orLtB (B) expressed in corn as measured by ELISA. Bars represent thestandard error of the mean. The number of animals sampled in each group(N) is indicated for each group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0030] By use of the term “fish” herein is meant fin-fish, shellfish,and other aquatic animals. Fin-fish include all vertebrate fish, whichmay be bony or cartilaginous fish. The prime candidate fin-fish speciesfor receiving the vaccine of the invention are salmonid fish, includingsalmon and trout species, particularly coho salmon (Oncorhynchuskisutch), brook trout (Salvelinus fontinalis), brown trout (Salmotrutta), chinook salmon (Oncorhynchus tshawytscha), masu salmon(Oncorhynchus masou), pink salmon (Oncorhynchus gorbuscha), rainbowtrout (Oncorhynchus mykiss), Arctic charr (Salvelinus alpinus) andAtlantic salmon (Salmo salar). However, any other fish speciessusceptible to infectious disease may benefit, such as ornamental fishspecies, koi, goldfish, carp, catfish, yellowtail, sea bream, sea bass,pike, halibut, haddock, tilapia, turbot, wolffish, and so on.

[0031] Examples of shellfish include, but are not limited to clams,lobster, shrimp, crab and oysters. Other cultured aquatic animalsinclude, but are not limited to eels, squid and octopi.

[0032] A “plant-derived” recombinant amino acid sequence is an aminoacid sequence engineered to be expressed in a transgenic plant whosesequence is not endogenous to the plant.

[0033] An amino acid sequence of the invention is one which, whenadministered to a fish, results in an antigenic or immunogenic responsein the fish.

[0034] Antigens of organisms causing pathologies in fish and nucleotidesequences encoding such antigens have been administered to fish thathave been exposed by way of injection, immersion, spray, adding thevaccine directly to fish food, or gene transfer into fish cells. Forexample, U.S. Pat. No. 6,462,027 describes a method of contacting anisolated non-infectious polynucleotide encoding an immunogen with anaquatic animal. U.S. Pat. No. 6,180,614 describes introducing DNAplasmids encoding antigen-based vaccines by transfection into the fish.The promoter is one capable of directing expression in the fish. Thepatent specification notes that bacterially-expressed recombinantproteins can form inclusion bodies so that recovery of protein is low ornonexistent. Further, it indicates induction of an immune response mayrequire that the antigenic protein be correctly glycosylated and folded,which, they state, may not be accomplished in a cell other than ananimal cell. However, the inventors here have found that it is possibleto produce in a plant a correctly processed antigenic amino acidsequence that can cause an antigenic or immunogenic response whenadministered to fish.

[0035] The coding sequences of many amino acid sequences producing anantigenic or immunogenic response in fish (also referred to as an“antigen”) have been and are being sequenced, as there has been a greatinterest in producing vaccines using such genes. While specific examplesare set forth below to illustrate the principle of the invention usingcertain antigens, the invention is not limited to any particularantigen. Rather any amino acid sequence that produces an antigenic orimmune response in a fish can be used. In a preferred embodiment, anantigen of an organism causing pathologies in fish is used. Such anantigen is used to induce or enhance immunity, and the correspondingnucleotide sequence which encodes that antigen is useful in theinvention. A few of the numerous example of such sequences which havebeen isolated include the cDNA encoding structural protein-1 ofinfectious salmon anemia virus (ISAV) described in U.S. Pat. No.6,471,964, as well as those discussed in Tucker et al. (2000)“Assessment of DNA vaccine potential for juvenile Japanese flounderParalichthys olivaceus, through the introduction of reporter genes byparticle bombardment and histopathology” Vaccine 19(7-8):801-809;Corbeil et al. (1999) “Evaluation of the protective immunogenicity ofthe N, P, M, NV, G proteins of infectious hematopoietic necrosis virusin rainbow trout Oncorhynchus mykiss using DNA vaccines” Dis. Aquat.Organ 39(1):29-26; Nusbaum et al. (2002) “Protective immunity induced byDNA vaccination of channel catfish with early and late transcripts ofthe channel catfish herpes virus (IHV-1)” Vet Immunol. Immunopathol84(3-4):151-168; Clark et al. (1992) “Developmental expression ofsurface antigen genes in the parasitic cilate Ichtyophthiriusmultifiliis” Proc. Natl. Acad. Sci. 89(14):6363-6367; and Sato et al.(2000) “Expression of YAV proteins and vaccination against viral ascitesamong cultured juvenile yellowtail” Biosci. Biotechnol. Biochem.64(7):1494-1497.

[0036] Examples of the variety of pathogens for which the methods of theinvention can be useful include, without limitation, hemorrhagicsepticemia virus (VHSV), infectious pancreatic necrosis virus (IPNV),infectious haematopoietic necrosis virus (IHNV), salmon pancreas diseasevirus (SPDV), virus causing spring viremia of carp, grass carphemorrhagic virus, nodaviridae such as nervous necrosis virus or stripedjack nervous necrosis virus, infectious salmon anaemia virus (ISAV),Aeromonis salmonicida, Renibacterium salmoninarum, Yersinia spp.,Pasteurella spp. (including Photobacterium damselae), Vibrio spp.(including V. anguillarum and V. ordalii), Edwardsiella spp. (includingE. ictaluri and E. tarda), Piscirickettsia salmonis (causative ofSalmonid Rickettsial Septicaemia), Iridovirus, cardiomyopathy syndromevirus, taura syndrome virus, Penaeus monodon virus, shrimp yellowheadvirus, shrimp whitespot virus, and Streptococci spp.

[0037] Other examples of known antigens that produce pathology in fishthat can be used in the invention include: IPNV VP2 and VP3 proteins,IHNV G protein, VHSV G protein, Nodavirus capsid protein, ISAV antigensdisclosed in WO 01/10469, SPDV antigens disclosed in WO 99/58639, P.salmonis antigens disclosed in WO 01/68865, and Whitespot Virus antigensdisclosed in WO 01/09340. Numerous nucleic acid and amino acid sequencesof fish pathogen antigens are known and accessible through the Genbankdatabases and other sources.

[0038] An amino acid sequence or antigen of the invention which is “ofan organism causing disease or pathology in fish” is an amino acidsequence or antigen of a pathogen of fish (or a derivative thereof),which is expressed in plant cells through recombinant DNA technology, asdescribed below. The “antigens” used in practicing the invention may befull-length antigenic proteins from a virus, bacterium, fungus,parasite, protozoan, etc., that causes disease in fish, or alternativelymay constitute an immunogenic portion, fragment or derivative of same. A“derivative” of an amino acid sequence is a sequence related to thereference sequence either on the amino acid sequence level or at the 3Dlevel (i.e. molecules having approximately the same shape andconfiguration as the reference sequence). Derivatives include sequencehomologues, mutants, mimetics, mimotopes, analogues, monomeric forms andfunctional equivalents whether obtained directly from the organism orsynthetically produced, which are capable of inducing an antigenic orimmunogenic response in fish. Particular mention may be made ofderivatives resulting from amino acid substitutions (with natural orsynthetic amino acids), deletions, inversions, insertions, andadditions.

[0039] This antigen, whether it is an amino acid sequence or protein, isthe “antigen of interest”. The “gene of interest” refers to thenucleotide sequence that encodes for the polypeptide or protein that isthe desired antigen or selection marker. The gene of interest can beoptimized for plant transcription and translation by optimizing thecodons used for plants (see discussion below).

[0040] In general, the methods available for construction of recombinantgenes described above, optionally comprising various modifications forimproved expression, can differ in detail. However, conventionallyemployed methods include PCR amplification, or the designing andsynthesis of overlapping, complementary synthetic oligonucleotides,which are annealed and ligated together to yield a gene with convenientrestriction sites for cloning. The methods involved are standard methodsfor a molecular biologist Sambrook et al., Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory Press, Second Edition(1989).

[0041] Once the gene is engineered to contain desired features, such asthe desired localization sequences, it is placed into an expressionvector by standard methods. The selection of an appropriate expressionvector will depend upon the method of introducing the expression vectorinto host cells. A typical expression vector contains prokaryotic DNAelements coding for a bacterial origin of replication and an antibioticresistance gene to provide for the growth and selection of theexpression vector in the bacterial host; a cloning site for insertion ofan exogenous DNA sequence, which in this context would code for theantigen of interest; eukaryotic DNA elements that control initiation oftranscription of the exogenous gene, such as a promoter; and DNAelements that control the processing of transcripts, such astranscription termination/polyadenylation sequences. It also can containsuch sequences as are needed for the eventual integration of the vectorinto the plant chromosome.

[0042] In a preferred embodiment, the expression vector also contains agene encoding a selection marker that is functionally linked to apromoter that controls transcription initiation. By “functionallylinked” it is understood that the gene of interest (in this case thegene encoding a selection marker) is down-stream of the promoter in thecorrect orientation and in the correct frame alignment such thattranscription of mRNA and translation of the mRNA occurs correctly toproduce the desired polypeptide or protein. For a general description ofplant expression vectors and reporter genes, see Gruber et al. (1993)“Vectors for Plant Transformation” in Methods of Plant Molecular Biologyand Biotechnology CRC Press. p 89-119. In one embodiment, the selectivegene is a glufosinate-resistance encoding DNA and in another embodimentcan be the phosphinothricin acetyl transferase (“pat”) or maizeoptimized pat gene under the control of the CaMV 35S promoter. The geneconfers resistance to bialaphos (Gordon-Kamm (1990) The Plant Cell 2:603; Uchimiya et al. (1993) Bio/Technology 11: 835; and Anzai et al.(1989) Mol. Gen. Gen. 219: 492).

[0043] By “promoter” is meant minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell-type specific, tissue-specific, orinducible by external signals or agents; such elements may be located inthe 5′ or 3′ regions of the gene. Although the endogenous promoter of astructural gene of interest may be utilized for transcriptionalregulation of the gene, the promoter is often a foreign regulatorysequence. Promoter elements employed to control expression of antigenicproteins and the selection gene, respectively, can be anyplant-compatible promoter. Those can be plant gene promoters, such as,for example, the ubiquitin promoter (European patent application no. 0342 926); the promoter for the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi, et al.,EMBO J., 3:1671, 1984; Broglie, et al., Science, 224:838, 1984); orpromoters from the tumor-inducing plasmids from Agrobacteriumtumefaciens, such as the nopaline synthase and octopine synthasepromoters (carried on tumor-inducing plasmids of Agrobacteriumtumefaciens and have plant activity); or viral promoters such as thecauliflower mosaic virus (CaMV) 19S and 35S promoters of CaMV (Brisson,et al., Nature, 310:511, 1984; Odell, et al., Nature, 313:810, 1985),the figwort mosaic virus 35S promoter(Gowda, et al., J. Cell Biochem.,13D: 301, 1989) or the coat protein promoter of TMV (Takamatsu, et al.,EMBO J. 6:307, 1987. See also Kay et al. (1987) “Duplication of CaMV 35Spromoter sequences creates a strong enhancer for plant genes” Science236:199-1302 and European Patent Application EP-A-342 926.Alternatively, plant promoters such as the mannopine synthase promoter(Velten, et al., EMBO J., 3:2723, 1984); heat shock promoters, e.g.,soybean hspl7.5-E or hspl 7.3-B (Gurley, et al., Mol. Cell. Biol.,6:559, 1986; Severin, et al., Plant Mol. Biol., 15:827, 1990); orethanol-inducible promoters (Caddick et al., Nature Biotech., 16:177,1998) may be used. See International Patent Application No. WO 91/19806for a review of illustrative plant promoters suitably employed in thepresent invention. In one embodiment of the present invention, the aminoacid-encoding DNA is under the transcriptional control of PGNpr6promoter (WO 01/94394). This is a ubiquitin-like promoter.

[0044] In a preferred embodiment, a tissue specific promoter is providedto direct transcription of the DNA preferentially to the seed. One suchpromoter is the globulin promoter. This is the promoter of the maizeglobulin-1 gene, described by Belanger, F. C. and Kriz, A. L. (1991)“Molecular basis for allelic polymorphism of the maize globulin-1 gene”Genetics 129: 863-972. It also can be found as accession number L22344in the Genbank database. Another example is the phaseolin promoter. See,Bustos et al. (1989) “Regulation of B-glucuronidase expression intransgenic tobacco plants by an A/T-rich cis-acting sequence foundupstream of a french bean B-phaseolin gene”, The Plant Cell (1):839-853.

[0045] The expression vector can optionally also contain a signalsequence located between the promoter and the gene of interest. A signalsequence is a nucleotide sequence, and possibly the corresponding aminoacid sequence, which is used by a cell to direct the protein orpolypeptide of interest to be translated and placed in a particularplace within or outside the eukaryotic cell. One example of a plantsignal sequence is the barley α-amylase secretion signal (Rogers, (1985)J. Biol Chem 260, 3731-3738). Many signal sequences are known in theart. See, for example Becker et al. (1992), Plant Mol. Biol. 20:49;Close, P. S., (1993) Master's Thesis, Iowa State University; Knox, C.(1987), et al., “Structure and Organization of Two DivergentAlpha-Amylase Genes from Barley”, Plant Mol. Biol. 9:3-17; Lerner etal., (1989) Plant Physiol. 91:124-129; Fontes et al. (1991), Plant Cell3:483-496; Matsuoka et al. (1991), Proc. Natl. Acad. Sci. 88:834; Gouldet al. (1989), J. Cell. Biol. 108:1657; Creissen et al. (1991), Plant J.2:129; Kalderon, et al. (1984) “A short amino acid sequence able tospecify nuclear location” Cell 39:499-509; and Steifel, et al. (1990)“Expression of a maize cell wall hydroxyproline-rich glycoprotein genein early leaf and root vascular differentiation” Plant Cell 2:785-793.

[0046] In one embodiment, the plant selection marker and the gene ofinterest can be both functionally linked to the same promoter. Inanother embodiment, the plant selection marker and the gene of interestcan be functionally linked to different promoters. In yet a third andfourth embodiments, the expression vector can contain two or more genesof interest that can be linked to the same promoter or differentpromoters.

[0047] Obviously, many variations on the promoters, selectable markers,signal sequences and other components of the construct are available toone skilled in the art.

[0048] In accordance with the present invention, a transgenic plant isproduced that contains a DNA molecule, comprised of elements asdescribed above, integrated into its genome so that the plant canexpress the gene of interest and thus produce the antigen of interest.The transgenic plant may suitably be a species that is conventionallycultivated for animal feed, such as corn (Zea mays), canola (Brassicanapus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), potato (Solanum tuberosum), tomatoes(Lycopersicon esculentum), and peas (Lathyrus spp.). Alternatively, thetransgenic plant may be a species that is not conventionally eaten, suchas tobacco (Nicotiana tabacum), cotton (Gossypium hirsutum), tea(Camellia sinensis), flax (Linum), sisal (Agave spp., Furcraea spp.),pines, firs and cedars. In order to create such a transgenic plant, theexpression vectors containing the gene can be introduced intoprotoplasts, into intact tissues, such as immature embryos andmeristems, into callus cultures, or into isolated cells. Preferably,expression vectors are introduced into intact tissues. General methodsof culturing plant tissues are provided, for example, by Miki et al.(1993) “Procedures for Introducing Foreign DNA into Plants” in Methodsin Plant Molecular Biology and Biotechnology, Glick et al (eds) CRCPress pp. 67-68 and by Phillips et al. (1988) “Cell/Tissue Culture andIn Vitro Manipulation” in Corn and Corn Improvement 3d Edit. Sprague etal (eds) American Soc. of Agronomy pp. 345-387. The selectable markerincorporated in the DNA molecule allows for selection of transformants.

[0049] Methods for introducing expression vectors into plant tissueavailable to one skilled in the art are varied and will depend on theplant selected. Procedures for transforming a wide variety of plantspecies are well known and described throughout the literature. See, forexample, Miki et al, supra; Klein et al. (1992) Bio/Technology 10:26;and Weisinger et al. (1988) Ann. Rev. Genet. 22: 421-477. For example,the DNA construct may be introduced into the genomic DNA of the plantcell using techniques such as microprojectile-mediated delivery (Kleinet al. (1987) Nature 327: 70-73); electroporation (Fromm et al. (1985)Proc. Natl. Acad. Sci. 82: 5824); polyethylene glycol (PEG)precipitation (Paszkowski et al. (1984) Embo. J. 3: 2717-272); directgene transfer (WO 85/01856 and EP-A-275 069); in vitro protoplasttransformation (U.S. Pat. No. 4,684,611) and microinjection of plantcell protoplasts or embryogenic callus (Crossway, (1985) Mol. Gen.Genetics 202:179-185). Co-cultivation of plant tissue with Agrobacteriumtumefaciens is another option, where the DNA constructs are placed intoa binary vector system (Ishida et al. (1996) “High efficiencytransformation of maize (Zea mays L.) mediated by Agrobacteriumtumefaciens” Nature Biotechnology 14:745-750). The virulence functionsof the Agrobacterium tumefaciens host will direct the insertion of theconstruct into the plant cell DNA when the cell is infected by thebacteria. See, for example Horsch et al. (1984) Science 233: 496-498,and Fraley et al. (1983) Proc. Natl. Acad. Sci. 80: 4803.

[0050] Standard methods for transformation of canola are described byMoloney et al. (1989) “High Efficiency Transformation of Brassica napusUsing Agrobacterium Vectors” Plant Cell Reports 8:238-242. Corntransformation is described by Fromm et al. (1990) Bio/Technology 8:833and Gordon-Kamm et al, supra. Agrobacterium is primarily used in dicots,but certain monocots such as maize can be transformed by Agrobacterium.See for example, U.S. Pat. No. 5,550,318. Rice transformation isdescribed by Hiei et al. (1994) “Efficient transformation of rice (Oryzasativs L.) mediated by Agrobacterium and sequence analysis of theboundaries of the T-DNA” The Plant Journal 6(2): 271-282, Christou etal. (1992) Trends in Biotechnology 10:239 and Lee et al. (1991) Proc.Nat. Acad. Sci. USA 88:6389. Wheat can be transformed by techniquessimilar to those used for transforming corn or rice. Sorghumtransformation is described by Casas et al. (1997) “Transgenic sorghumplants obtained after microprojectile bombardment of immatureinflorescences” In vitro cellular and developmental biology, Plant.33:92-100 and by Wan et al. (1994) Plant Physiology. 104:37. Soybeantransformation is described in a number of publications, including U.S.Pat. No. 5,015,580.

[0051] In one preferred method, the Agrobacterium transformation methodsof Ishida supra and also described in U.S. Pat. No. 5,591,616, aregenerally followed, with modifications that the inventors have foundimprove the number of transformants obtained. The Ishida method uses theA188 variety of maize that produces Type I callus in culture. In onepreferred embodiment the Hi II maize line is used which initiates TypeII embryogenic callus in culture. While Ishida recommends selection onphosphinothricin when using the bar or pat gene for selection, anotherpreferred embodiment provides for use of bialaphos instead. In general,as set forth in the '616 patent, and as outlined in more detail below,dedifferentiation is obtained by culturing an explant of the plant on adedifferentiation-inducing medium for not less than seven days, and thetissue during or after dedifferentiation is contacted with Agrobacteriumhaving the gene of interest. The cultured tissue can be callus, anadventitious embryo-like tissue and suspension cells, for example. Inthis preferred embodiment, the suspension of Agrobacterium has a cellpopulation of 10⁶ to 10¹¹ cells/ml and are contacted for three to tenminutes with the tissue, or continuously cultured with Agrobacterium fornot less than seven days. The Agrobacterium can contain plasmid pTOK162,with the gene of interest between border sequences of the T region ofthe plasmid, or the gene of interest may be present in anotherplasmid-containing Agrobacterium. The virulence region may originatefrom the virulence region of a Ti plasmid or Ri plasmid. The bacterialstrain used in the Ishida protocol is LBA4404 with the 40 kb superbinary plasmid containing three vir loci from the hypervirulent A281strain. The plasmid has resistance to tetracycline. The cloning vectorcointegrates with the super binary plasmid. Since the cloning vector hasan E. coli specific replication origin, but not an Agrobacteriumreplication origin, it cannot survive in Agrobacterium withoutcointegrating with the super binary plasmid. Since the LBA4404 strain isnot highly virulent, and has limited application without the superbinary plasmid, the inventors have found in yet another embodiment thatthe EHA101 strain is preferred. It is a disarmed helper strain derivedfrom the hypervirulent A281 strain. The cointegrated superbinary/cloning vector from the LBA4404 parent is isolated andelectroporated into EHA 101, selecting for spectinomycin resistance. Theplasmid is isolated to assure that the EHA101 contains the plasmid.EHA101 contains a disarmed pTi that carries resistance to kanamycin.Hood E E, Helmer G L, Fraley R T, Chilton M D (1986) “The hypervirulenceof Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542outside of T-DNA” J. Bacteriol 168: 1291-1301.

[0052] Further, the Ishida protocol as described provides for growingfresh culture of the Agrobacterium on plates, scraping the bacteria fromthe plates, and resuspending in the co-culture medium as stated in the'616 patent for incubation with the maize embryos. This medium includes4.3 g MS salts, 0.5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride,1.0 ml thiamine hydrochloride, casamino acids, 1.5 mg 2,4-D, 68.5 gsucrose and 36 g glucose, all at a pH of 5.8. In a further preferredmethod, the bacteria are grown overnight in a 1 ml culture, then a fresh10 ml culture re-inoculated the next day when transformation is tooccur. The bacteria grow into log phase, and are harvested at a densityof no more than OD600=0.5 , preferably between 0.2 and 0.5. The bacteriaare then centrifuged to remove the media and resuspended in theco-culture medium. Since Hi II is used, medium preferred for Hi II isused. This medium is described in considerable detail by Armstrong, C.I. and Green C. E. (1985) “Establishment and maintenance of friable,embryogenic maize callus and involvement of L-proline” Planta154:207-214. The resuspension medium is the same as that describedabove. All further Hi II media are as described in Armstrong et al. Theresult is redifferentiation of the plant cells and regeneration into aplant. Redifferentiation is sometimes referred to as dedifferentiation,but the former term more accurately describes the process where the cellbegins with a form and identity, is placed on a medium in which it losesthat identity, and becomes “reprogrammed” to have a new identity. Thusthe scutellum cells become embryogenic callus.

[0053] It is preferred to select the highest level of expression of theamino acid sequence, and it is thus useful to ascertain expressionlevels in transformed plant cells, transgenic plants and tissue specificexpression. One such method is to measure the expression of the antigenof interest as a percentage of total soluble protein. One standard assayis the Bradford assay which is well known to those skilled in the art(Bradford, M. (1976) Anal. Biochem. 72:248). The biochemical activity ofthe recombinant amino acid sequence should also be measured and comparedwith a wild-type standard.

[0054] The levels of expression of the gene of interest can be enhancedby the stable maintenance of the gene of interest on a chromosome of thetransgenic plant. Use of linked genes, with herbicide resistance inphysical proximity to the gene of interest, would allow for maintainingselective pressure on the transgenic plant population and for thoseplants where the genes of interest are not lost.

[0055] With transgenic plants according to the present invention, theamino acid sequence can be produced in commercial quantities. Thus, theselection and propagation techniques described above yield a pluralityof transgenic plants that are harvested in a conventional manner. Theplant seed expressing the recombinant amino acid sequence can be used ina commercial process, or the amino acid sequence can be extracted. Whenusing the seed itself, it can, for example, be made into flour and thenapplied in the commercial process. Extraction from biomass can beaccomplished by known methods. Downstream processing for any productionsystem refers to all unit operations after product synthesis, in thiscase protein production in transgenic seed (Kusnadi et al. (1997)Biotechnology and bioengineering. 56:473-484). Seed is processed eitheras whole seed ground into flour, or fractionated, and the germ separatedfrom the hulls and endosperm. If germ is used, it is usually defattedusing a hexane extraction and the remaining crushed germ ground into ameal or flour. In some cases the germ is used directly or the amino acidsequence can be extracted (See, e.g. WO 98/39461). Extraction isgenerally made into aqueous buffers at specific pH to enhancerecombinant amino acid sequence extraction and minimize native seedprotein extraction. Subsequent amino acid sequence concentration orpurification can follow.

[0056] In a further embodiment, plant breeding can be used to introducethe gene into other plants once transformation has occurred. This can beaccomplished by any means known in the art for breeding plants such as,for example, cross pollination of the transgenic plants that aredescribed above with another plant, and selection for plants fromsubsequent generations which express the amino acid sequence. The plantbreeding methods used herein are well known to one skilled in the art.For a discussion of plant breeding techniques, see Poehlman (1987)Breeding Field Crops, AVI Publication Co., Westport Conn. Many cropplants useful in this method are bred through techniques that takeadvantage of the plant's method of pollination. A plant isself-pollinating if pollen from one flower is transferred to the same oranother flower of the same plant. A plant is cross-pollinated if thepollen comes from a flower on a different plant. For example, inBrassica, the plant is normally self sterile and can only becross-pollinated unless, through discovery of a mutant or throughgenetic intervention, self compatibility is obtained. Inself-pollinating species, such as rice, oats, wheat, barley, peas,beans, soybeans, tobacco and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower. Maize plants (Zea mays L.) can be bred byboth self-pollination and cross-pollination techniques. Maize has maleflowers, located on the tassel, and female flowers, located on the ear,on the same plant. It can self or cross pollinate.

[0057] Pollination can be by any means, including but not limited tohand, wind or insect pollination, or mechanical contact between the malefertile and male sterile plant. For production of hybrid seeds on acommercial scale in most plant species pollination by wind or by insectsis preferred. Stricter control of the pollination process can beachieved by using a variety of methods to make one plant pool malesterile, and the other the male fertile pollen donor. This can beaccomplished by hand detassling, cytoplasmic male sterility, or controlof male sterility through a variety of methods well known to the skilledbreeder. Examples of more sophisticated male sterility systems includethose described at Brar et al., U.S. Pat. Nos. 4,654,465 and 4,727,219and Albertsen et al. U.S. Pat. Nos. 5,859,341 and 6,013,859.

[0058] Backcrossing methods may be used to introduce the gene into theplants. This technique has been used for decades to introduce traitsinto a plant. An example of a description of this and other plantbreeding methodologies that are well known can be found in referencessuch as Plant Breeding Methodology edit. Neal Jensen, John Wiley & Sons,Inc. (1988). In a typical backcross protocol, the original variety ofinterest (recurrent parent) is crossed to a second variety (nonrecurrentparent) that carries the single gene of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a plant is obtainedwherein essentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

[0059] The preferred method of administration of plant-derivedrecombinant amino acid sequence to fish is per oral, optionally byadmixture of the recombinant amino acid sequence to a conventionalfeedstuff. Alternative methods of administration include immersion,intra-peritoneal injection, and intramuscular injection.

[0060] Transgenic plant tissue may be fed to the fish, or mixed withother materials and fed to fish, or extracted and administered to thefish.

[0061] Oral delivery forms of the vaccine encompass any combination ofthe recombinant amino acid sequence with one or more excipients andoptionally with one or more nutrients. Excipients as used herein caninclude silica, binding agents, emulsions, tensio-active substances,fatty acids, fats, oils etc. and any other additives necessary forpreparing the composition.

[0062] Typical fish feedstuffs can comprise various nutrient sources,such as a metabolizable energy source (carbohydrate), a protein source,a fat source, and optionally fibers, vitamins and minerals. The exactcomposition of the feedstuff depends on the type of fish concerned, andin particular whether or not the fish are carnivorous. On a commercialscale feedstuffs may conveniently be provided in the form of pressed orextruded feed pellets. Plant-derived recombinant amino acid sequence maybe incorporated into the feed by substitution for a more usual proteinsource (such as fish meal, blood meal, maize gluten, soya meal etc.).Alternatively, the plant-derived recombinant amino acid sequence may beadhered to the surface of a pre-formed fish feedstuff.

[0063] The plant-derived recombinant amino acid sequence may beenteric-coated for oral delivery. The enteric coating protects thevaccine from proteases and from the relatively low pH levels of thestomach. This allows the vaccine to reach the hindgut associated withlymphoid tissue, which maximizes the effectiveness of the vaccine forprotecting fish. The enteric coating typically comprises a polymercoating that is unaffected by acidic pH, but which is dissolved uponpassing to the higher pH environments of the intestine.

[0064] In a preferred embodiment the plant-derived recombinant aminoacid sequence is administered to fish in the form of transgenic plantmaterial, such as plant seeds, leaves, fruits, stems, tubers, etc.,preferably where the transgenic plant material is not admixed to anyother feedstuffs. In another embodiment the plant-derived recombinantamino acid sequence is physically (reversibly) mixed with pre-formedfish feed immediately prior to feeding the fish.

[0065] In order to avoid unnecessary extraction procedures, it ispreferred to deliver the plant-derived recombinant amino acid sequencein a non-purified (crude) form to the fish. This means that edible partsof the source plant are not specially treated or processed in order toextract or concentrate the recombinant amino acid sequence.

[0066] The effective dosage of vaccine may vary depending on the sizeand species of the subject, and according to the mode of administration.The optimal dosage can be determined through trial and error by aveterinarian or aquaculture specialist. Vaccines may comprise betweenabout 1 and 1000 μg, preferably between about 10 and 200 μg, morepreferably between about 50 and 100 μg of recombinant amino acidsequence in a single dosage.

[0067] The vaccine of the invention may be administered to fish forprophylactic or therapeutic purposes. The vaccine is capable of inducinglong term protection against the target infectious disease. “Long term”protection in the case of fish means a protective immune response forlonger than 7 days, more preferably longer than 20 days, and mostpreferably longer than 70 days post vaccination.

EXAMPLE 1 Transformation of Avidin into Plants and Detection ofExpression Levels

[0068] Construction of Plasmids for Avidin Expression in Plants

[0069] Construction of plasmids for avidin transformation into corn isdescribed in U.S. Pat. No. 5,767,379, incorporated herein by reference.The chicken egg white avidin cDNA was reported by Gope M L. (1987), etal., Nuc. Acids Res. 15: 3595-3606. The amino acid sequence is reversetranslated into nucleic acid sequence utilizing a preferred maize codonusage table (GCG, assembled by Mike Cherry, Stanford University). Fromthis computer-generated synthetic sequence, overlapping, complementaryoligonucleotides with compatible restriction site termini are designed,then annealed and ligated to yield the maize optimized gene. Thesequence used is set forth in the '379 patent, incorporated byreference. The barley alpha amylase signal sequence ((Rogers, (1985) J.Biol Chem 260, 3731-3738) is also synthesized (using overlapping,complementary nucleotides) with maize-preferred codons. Compatiblerestriction sites between these two gene fragments are ligated, with thebarley alpha amylase signal sequence at the 5′ end of the avidin geneand in proper frame alignment so that the correct codon usage occursduring translation to yield the desired antigen. The resultant barleyalpha amylase signal sequence/avidin segment is cloned, (See FIG. 1 (SEQID NO: 1)) as a BamHI/EcoRI fragment, into the vector pGEM3Zf+, aproduct of Promega Corporation (Madison, Wis.), to generate plasmidpPHI5142. A BamHI/HpaI fragment containing the barley alpha amylasesignal sequence/avidin region is isolated and cloned into a plasmidderived from pBlueScript SK+ (Stratagene, La Jolla, Calif.), as abackbone. In this plasmid, the signal sequence/avidin gene fragment isinserted, in the correct orientation, between the maize ubiquitin 5′region, which includes the maize ubiquitin promoter (UBI1ZM), the firstexon and first intron, and the potato proteinase inhibitor II (PinII)transcription terminator region (An et al, (January 1989 (Plant Cell1:115-122). The resultant plasmid is pPHI5168 (FIG. 2). Co-transformedwith the plasmid is a plasmid (pPHI610) containing the bar gene fromStreptomyces hygroscopicus, supra and White J. (1990) Nucleic Acids Res18:1062 linked to the double 35S promoter (e.g. Friz, S. E. J. Cell Sci98:545-550), the intron from the maize alcohol dehydrogenase gene(Callis J., et al. Genes and Development 1:1183-1200) and the PinIIterminator (An G., et al. (1989) Plant Cell 1:115-122). These constructsand the process used are fully described in the '379 patent, supra. Notethat in the experiment described in the '379 patent, the bar gene isused, where in the other experiments described herein the maizeoptimized pat gene is used. FIG. 3 sets forth this sequence (SEQ ID NO:2).

[0070] Transformation and Tissue Culture to Produce Avidin-ExpressingPlants.

[0071] An established callus line derived from a single immature embryoof the “Hi II” maize plants (Armstrong C L, Green C E, Phillips R L(1991) Maize Gen. Coop. Newsletter, 65:92-93) is transformed usingparticle bombardment-mediated transformation with a helium-poweredparticle acceleration device, PDS 1000 (Bio-Rad, Hercules, Calif.). HiII is a corn plant line used in research frequently because of its easein transformation. Tissue showing a friable type-II embryogenicmorphology is sieved through 710 m mesh prior to co-transformation withequimolar amounts of the avidin gene (pPHI5168) and the bar selectablemarker gene (PHP610), according to the procedures of Tomes et al. (TomesD T, Ross M C, Songstad D D (1995) Plant Cell Tissue and Organ Culture:Fundamental Methods. Springer-Verlag, Berlin, Heidelberg. pp.197-213).Transformants expressing the bar gene are selected in the presence ofbialaphos (3 mg l⁻¹), according to the protocol of Register et al.(Register J. C.-III et al. (1994) , Plant Mol. Biol. 25:951-961).Co-transformants that also express the avidin gene are identified byELISA screening of the selected colonies. Multiple plants (T₀generation) are regenerated from avidin-expressing colonies, transferredto the greenhouse and assayed for avidin expression in leaf tissue.) T₁seed is obtained by outcrossing, with the T₀ plants as the female parentand a non-transformed inbred line (PHN46; see U.S. Pat. No. 5,567,861)as the male parent.

[0072] ELISA to Detect Avidin in Corn.

[0073] The following procedures are used to detect expression of avidinin seeds. Seeds are powdered and extracted in 10 mM PBS pH 7.0containing 0.05% Tween-20 (PBST). Total protein was quantified using theBradford microtiter assay Bradford (Bradford, M. M. 1976. A rapid andsensitive method for the quantitation of microgram quantities of proteinutilizing the principal of protein-dye binding. Anal. Biochem.72:248-254). ELISAs are typical sandwich style in which the microtiterplates are coated with rabbit anti-avidin antibody, the avidin proteinis captured overnight at 4° C., and the plate is reacted with goatanti-avidin antibody (Vector Labs, Burlingame, Calif.) followed byanti-goat alkaline phosphatase conjugate (Jackson Immunoresearch, WestGrove, Pa.). The alkaline phosphatase is detected with para-nitrophenylphosphate and read at 405 nm on a SpectroMax plate reader (MolecularDevices, Sunnyvale, Calif.).

EXAMPLE 2 Transformation of LtB into Plants and Detection of Expression

[0074] LtB sequences and introduction into plants is described at U.S.Pat. No. 6,194,560, which sequences and methods were used in thisexperiment, and which is incorporated herein by reference. The vectorused here differs in certain aspects from that described in the '560patent. It is PGN7101, shown in FIG. 4. The LtB gene of an E. colistrain of human origin (Leong et al.(1985) Nucleotide sequencecomparison between heat-labile toxin B-subunit cistrons from Escherichiacoli of human and porcine origin Infect Immun. April; 48(1):73-7) issynthesized to optimize codon usage for maize, see FIG. 5A (SEQ ID NO:3). Oligonucleotides spanning the gene are annealed and ligated, and theproducts are amplified using the polymerase chain reaction (PCR). Anoligonucleotide sequence encoding the barley α-amylase secretion signal(BAASS) is added at the N-terminus of LtB using PCR and this completeBAASS:LtB sequence fragment is inserted into a vector backbone resultingin the plasmid PGN543 1. The BAASS:LtB sequence is shown in FIG. 5B (SEQID NO: 4). The BAASS:LtB sequence is removed from PGN5431 using therestriction enzymes NcoI and HpaI and ligated into the correspondingrestriction sites in the vector PGN2774 resulting the intermediatevector PGN7020. In this intermediate vector, the BAASS:LtB is placed 3′to a maize constitutive promoter and untranslated leader sequence fromthe ubiquitin regulatory system, designated PGNpr1 (wild type maizepolyubiquitin-1), and 5′ to the potato proteinase inhibitor IItranscription terminator (PinII). The BAASS:LtB expression cassette(promoter, leader, BAASS:LtB and Pin II sequences) is removed fromPGN7020 using the restriction enzymes NheI and NotI and ligated into thecorresponding sites in the plant transformation vector PGN3770. Thefinal BAASS:LtB transformation vector, designated PGN7101, contains theright and left border sequences of Agrobacterium tumefaciens Ti plasmidorigin, and the pat gene of Streptomyces viridichromogenes, conferringresistance to glufosinate ammonium.

EXAMPLE 3 Transformation of IPNV into Plants and Detection of Expression

[0075] Infectious pancreatic necrosis virus (IPNV) infects mollusks,crustaceans and many types of fish, especially salmonids. IPNV infectioncan have devastating effects on salmonid production due to fishmortality at the fry or smolt stage and decreased growth in survivingpopulations. There have been many attempts to produce an effectivevaccine against this virus. So far protection has been seen only with aninjected inactivated virus, however this vaccine has proven to beexpensive and impractical. The major structural and immunogenic proteinsof the virus, VP2 and VP3, are expressed in maize using the methodsdescribed, supra.

[0076] Nucleotide sequences for VP2 and VP3 are initially obtained fromthe plasmids pUK-NVP2 and pUK-NVP3 respectively. The sequences for theproteins in these two plasmids are from a Norwegian IPNV strain closelyrelated to the NI strain. Some nucleotide modification is carried out onthe 5′ and 3′ ends of the gene sequences to optimize codon usage formaize.

[0077] An oligonucleotide sequence encoding 5′ VP2 sequences, that aremissing from the VP2 gene in pUK-NVP2, along with nucleotide changes forcodon optimization, is annealed at the 5′ end of the VP2 sequence frompUK-NVP2 using polymerase chain reaction (PCR). An oligonucleotidesequence encoding nucleotide changes for codon optimization at the 3′end of VP2 along with sequences from the potato proteinase inhibitor IItranscription terminator (PinII) (An et al., Plant Cell (1989)1:115-122) is added at the 3′ end of the VP2 sequence from pUK-NVP2using PCR. These two PCR fragments along with an internal VP2 fragment,isolated using the restriction enzymes SacII and BbsI, from pUK-NVP2 areligated together to give the plasmid PGNK5676 containing the completeVP2 nucleotide sequence (SEQ ID NO: 5) shown in FIG. 6. Anoligonucleotide sequence encoding the barley α-amylase secretion signal(BAASS) is added at the N-terminus of the restored VP2 gene using PCR.The fragment generated from PCR is put into a vector backbone resultingin the plasmid PGNK5443 containing the BAASS:VP2 nucleotide sequence(SEQ ID NO: 6) shown in FIG. 7.

[0078] Oligonucleotides encoding nucleotide changes for codonoptimization for maize are annealed to both the 5′ and 3′ ends of theVP3 sequences, from pUK-NVP3, using PCR. The PCR fragment is put into avector backbone to give the plasmid PGNK5581 containing the partiallyoptimized VP3 sequence (SEQ ID NO: 7) shown in FIG. 8. Anoligonucleotide sequence encoding BAASS is added to the N-terminus ofVP3 using PCR. The PCR fragment is put into a vector backbone to givethe plasmid PGNK5330 containing the BAASS:VP3 sequence (SEQ ID NO: 8)shown in FIG. 9.

[0079] Two separate plant transformation vectors are constructed, eachcontaining both of the genes for VP2 and VP3. The first constructcontains the sequences for BAASS:VP2 and BAASS:VP3, each in a separateexpression cassette containing a maize seed preferred promoter,designated PGNpr2, and the PinII terminator. The BAASS:VP2 sequences arecut from PGNK5443 with the restriction enzymes NcoI and PacI. Thisfragment along with the PGNpr2 fragment cut with the restriction enzymesHindIII and NcoI are ligated into the HindIII and PacI restriction sitesof the PGN9004 plant transformation vector which contains the PinIIterminator, the right and left border sequences of Agrobacteriumtumefaciens Ti plasmid origin, and the pat gene of Streptomycesviridichromogenes, conferring resistance to glufosinate ammonium. Thisplasmid is designated PGNK5461. In a similar process the BAASS:VP3sequences are cut from PGNK5330 using NcoI and PacI. This fragment alongwith the HindIII/NcoI PGNpr2 fragment is ligated into the HindIII andPacI sites of PGN9004 resulting in the plasmid PGNK5335. The BAASS:VP2expression cassette, containing the PGNpr2 promoter, BAASS, VP2 and thePinII terminator, is cut from PGNK5461 using the restriction enzymesAscI and PacI. The BAASS:VP3 expression cassette, containing the PGNpr2promoter, BAASS, VP3 and the PinII terminator, is cut from PGNK5335using the restriction enzymes HindIII and MluI. These two fragments areligated into the HindIII and PacI restriction sites of PGN9004 resultingin the final plant transformation vector containing both the BAASS:VP2and BAASS:VP3 expression cassettes. This construct, designated PGN9084(FIG. 10), is designed such that the proteins are sent to the cell walland accumulate primarily in the seed. The plants are then transformedaccording to the modified Ishia protocol, set forth supra. Plantsresulting from the transformation of PGN9084 are designated NVA.

[0080] The second plant transformation vector also contains both VP2 andVP3 in separate expression cassettes under the control of the PGNpr2promoter and the PinII terminator, however the barley α-amylasesecretion signal (BAASS) is not present. A 5′ portion of the VP2sequence up to and including the BstBI restriction site is cut fromPGNK5573 using the restriction enzymes BbsI and BstBI. This fragment andthe HindIII/NcoI PGNpr2 fragment are ligated into the HindIII and BstBIsites in PGNK5461 resulting in the plasmid PGNK5676 containing the VP2expression cassette. The VP3 sequence is cut from PGNK5581 using therestriction enzymes NcoI and PacI. This fragment and the HindIII/NcoIPGNpr2 fragment are ligated into the HindIII and PacI sites of PGN9004resulting in the plasmid PGNK5681 containing the VP3 expressioncassette. The VP2 expression cassette is cut from PGNK5676 using therestriction enzymes AscI and PacI. The VP3 expression cassette is cutfrom PGNK5681 using the enzymes HindIII and MluI. These two fragmentsare ligated into the HindIII and PacI restriction sites of PGN9004resulting in the final plant transformation vector containing both theVP2 and VP3 expression cassettes. This construct, designated PGN9111(FIG. 11), is designed such that the proteins are sent to the cytoplasmand accumulate primarily in the seed. Plants resulting from thetransformation of PGN9111 are designated NVB.

[0081] Western blot analysis using polyclonal anti-IPNV whole virusantibodies shows expression of the proteins VP2 and VP3 in both NVA andNVB seed. The VP2 and VP3 proteins expressed in NVA seed run slightlylarger than the corresponding native proteins found in the IPNV wholevirus standard on a Western blot (FIG. 12). Lane 1 shows proteinmarkers, lanes 2-4 a purified prep of IPNV whole virus, lane 5 controlmaize seed extract, negative control, lanes 6-11 extracts from variousNVA seed and lane 12 an unpurified prep of IPNV whole virus. (Note, thepurification process of the whole virus generates the smearing patternin the top of those wells).

[0082] Since the VP2 and VP3 proteins are targeted by the BAASS to thecell wall in the NVA seed, it is expected that the proteins will beglycosylated. Not wishing to be bound by theory, it is possible thatthis increase in size of both proteins suggests glycosylation of theproteins in the plant. Both VP2 and VP3 expressed in NVB seed run at theexpected sizes compared to the IPNV whole virus standard on a Westernblot (FIG. 13). Lane 1 shows protein markers, lane 2 extract from NVAseed, lanes 3-9 extracts from various NVB seed, and lanes 10-12increasing amounts of unpurified IPNV whole virus.

[0083] Since these proteins are expressed in the cytoplasm of the plantcell, no modification of the proteins is expected.

[0084] Expression levels of VP2 and VP3 in the NVA seed are measured bymeans of a Western blot. The intensity of the VP2 and VP3 protein bandsfrom the seed extracts are measured using spot densitometry and are thencompared to the bands of known amounts of whole virus. Using this methodthe expression of VP2 in NVA T2 seed is calculated to be 0.1% TSP (totalsoluble protein) and the expression of VP3 in NVA T2 seed is calculatedto be 0.3% TSP. Expression levels of VP2 in the NVB seed are measured byELISA. The ELISA is a typical sandwich style in which the microtiterplate is coated with sheep anti-IPNV whole virus antiserum, the IPNVprotein in the plant extract is captured overnight at 4° C., and theplate is reacted with AS1 mouse monoclonal anti-VP2 antibody followed byalkaline phosphatase conjugated sheep anti-mouse IgG. The alkalinephosphatase is detected with para-nitrophenyl phosphate, disodium (pNpp)and read at 405 nm on an absorbance microplate reader. Using his methodthe expression of VP2 in NVB T1 seed is measured to be 0.17% TSP in thehighest single seeds.

EXAMPLE 4 Feeding Studies with Avidin and LtB

[0085] To evaluate this new technology in fish, this experiment isdesigned to determine if oral administration of diets containingcorn-expressed recombinant marker proteins induces a humoral immuneresponse in salmonids. Atlantic salmon are fed, in an amount ofapproximately 2% of body weight per day, diets containing two doses ofunpurified corn expressing LtB (5% or 10% of food) or chicken egg whiteavidin (10% or 20% of food) for 5 days, 12 days with normal food and 5days with the treated diet. Groups of fish are also intraperitoneallyinjected with purified LtB and avidin protein as positive controls.

[0086] Fish growth, persistence of recombinant proteins in feces andhumoral immune response are examined. Fish antibody response is comparedfor the different doses of LtB, which has been shown previously to becapable of producing a strong antibody response in mice, and avidin,which has been shown previously to be a weaker antigen in mice.

[0087] The Atlantic salmon weigh about 20 grams each. There are a totalof eleven treatment groups with several negative and positive controlgroups (Table 1). A and B positive control groups, each consisting often fish, are given an intraperitoneal injection with oil-adjuvantedpreparations with group A receiving a single injection of 4 μg LtBprotein per fish and group B receiving a single injection of 20 μgavidin protein per fish which are both recombinant proteins purifiedfrom a corn expression system. Nine groups each contain 35 fish, withgroup C to G being negative controls. Group C receives commercial fishpellets. Group D receives pellets which include 5% non-transgenic corngerm. Group E receives pellets with 10% non-transgenic corn germ. GroupF receives pellets made with fish meal having 10% non-transgenic cornflour and Group G receives pellets with 20% non-transgenic corn flour.In the experimental groups, Group H receives fish pellets with 5% LtBtransgenic corn germ, and group I receives pellets with 10% LtB corngerm. Group J receives pellets with 10% transgenic avidin-containingflour, and group K receives pellets with 20% avidin flour. TABLE 1 TotalWeight protein Feed Number (g) corn amount (g) for Group Vaccine of Fishrequired (mg) 10 days A Injected Lt-B positive 10 0 0.040* 40 control BInjected Avidin 10 0 0.20* 40 positive control C Negative Control 1 35 00 140 (normal food) D Negative Control 2 5% 35 0 0 140 normal corn germmeal E Negative Control 2 35 0 0 140 10% normal corn germ meal FNegative Control 4 35 14 0 140 10% normal corn flour G Negative Control5 35 28 0 140 20% normal corn flour H 5% recombinant LtB 35 7 2.1 140corn germ meal I 10% recombinant LtB 35 14 4.2 140 corn germ meal J 10%avidin corn flour 35 14 20.61 140 K 20% avidin corn flour 35 28 41.22140

[0088] Fish are maintained at 10° C. At two, four, seven, fourteen andtwenty one days post-feeding five fish are sacrificed in nine dietgroups C-K to measure the persistence of the recombinant proteins in thefeces using an ELISA. At eight weeks, ten fish in all eleven groups aresacrificed weighed and specific antibody in the serum is measured byELISA

[0089] ELISA to Detect Marker Proteins in Feces:

[0090] ELISA is the typical sandwich style in which the microtiterplates are coated overnight at 4° C. with rabbit anti-avidin or anti-LtBantibody. Fish fecal samples, diluted in PBST, are added to wells andthe plate incubated overnight at 4° C. to allow capture of the avidin orLtB protein. The plate is reacted with goat anti-avidin antibody (VectorLabs, Burlingame, Calif.) or mouse biotinylated LtB antibody followed byanti-goat alkaline phosphatase conjugate (Jackson Immunoresearch, WestGrove, Pa.) or ExtraAvidin-alkaline phosphatase (Sigma-Aldrich CanadaLtd., Oakdale, ON). The alkaline phosphatase is detected withpara-nitrophenyl phosphate (Pierce, distributor MJS Biolynx Inc.,Brockville, ON, Canada) and read at 405 nm on a plate reader (BioTekInstruments Inc., Vermont).

[0091] ELISA to Detect Specific Anti-Avidin and Anti-LtB Antibodies inFish Serum:

[0092] A sandwich ELISA is used to detect specific antibodies in fishserum. Plate wells are coated with purified LtB or purifiedcorn-expressed avidin (Sigma) overnight at 4° C., two-fold dilutions offish serum in PBST-1% BSA are added and the plates incubated overnightat 17° C. to allow fish antibody capture. Primary antibody, monoclonalanti-Atlantic salmon Ig (Cedarlane Laboratories Ltd., ON, Canada),followed by secondary alkaline-phosphatase labeled anti-mouse Igantibody (Cedarlane), both diluted in PBST-1% BSA, are added to theplates. After the addition of para-nitrophenyl phosphate substrate(Pierce), absorbance is read at 405 nm using a microtiter plate reader(BioTek Instruments). Antibody titer is calculated as the endpointdilution.

[0093] The addition of ground corn expressing the two marker proteins tothe diet does not affect fish growth as shown in FIG. 14. The two markerproteins are detectable for an extended time period in the feces, atleast 21 days after cessation of feeding the treated diet as shown inTable 2. TABLE 2 Day Post-Feeding Group 2 4 7 14 21  5% LtB 5/5 4/4 5/54/5 NS 10% LtB 5/5 4/4 3/4 3/4 2/4 10% avidin 5/5 5/5 4/5 5/5 4/4 20%avidin 3/3 4/4 5/5 4/4 4/4

[0094] Oral administration of the marker proteins induces a humoralimmune response. At 8 weeks post-vaccination, only fish in the negativecontrol groups do not have a detectable specific serum antibodyresponse. The antibody response of fish fed unpurified corn-expressedmarker proteins is as strong as those of fish injected with pureproteins in oil adjuvant as shown in FIG. 15.

EXAMPLE 5 Feeding Studies with IPNV

[0095] The methods described in feeding the corn containing infectiouspancreatic necrosis virus VP2 and VP3 proteins will be completed. Thepresence of the viral proteins in the feces and organs of the animal isexpected, as well as antibody responses After fish are challenged withvirulent virus it is expected that oral administration of thecorn-expressed IPNV proteins will result in protection.

[0096] Fish will be divided into seven groups and tagged foridentification. Positive control group A will consist of fish given aninjection with a commercial vaccine that induces protection against IPNVand negative control group B will be fed commercial food pellets. Theremaining groups will be fed food containing corn germ with and withoutexpressed IPNV proteins for 5 days, 12 days with normal food and 5 dayswith food containing corn germ as outlined in Table 3. The percentincorporation rate of corn germ into food (g corn per g food) will be10% and 20%. TABLE 3 Number of Group fish Treatment A 55 ip injectedcommercial vaccine B 55 normal food C 85 non-transgenic corn germ mixedinto food 20% incorporation D 85 NVA corn germ 10% incorporation E 85NVA corn germ 20% incorporation F 85 NVB corn germ 10% incorporation G85 NVB corn germ 20% incorporation

[0097] At 4 weeks post-vaccination, all fish will acclimated over a fewdays to salt water and then maintained in flowing salt water at ambienttemperature (9-12° C.).

[0098] At 5 weeks post-transfer to salt water, fish will be exposed to acohabitation IPNV challenge. Naive fish will be injected with live IPNVand added to tanks containing the vaccinated fish. Daily mortality willbe monitored for five weeks.

[0099] On a weekly basis from 1 to 5 weeks post-vaccination and at thetime of challenge, 5 fish per groups C to G will be sacrificed andsampled for feces and organs to examine IPNV protein persistence anddistribution. Fish killed at the time of challenge, including 5 fish ingroups A and B, will also be bled and the serum tested for antibodies byELISA.

1 8 1 459 DNA Hordeum vulgare 1 atggccaaca agcacctgag cctctccctcttcctcgtgc tcctcggcct ctccgcctcc 60 ctcgccagcg gcgccaggaa gtgctccctcaccggcaagt ggaccaatga cctcggctcc 120 aacatgacca tcggcgccgt gaactccaggggcgagttca ccggcaccta catcaccgcc 180 gtgaccgcca cctccaacga gatcaaggagtcccccctcc acggtaccca gaacaccatc 240 aacaagagga cccagcccac cttcggcttcaccgtgaact ggaagttctc cgagtccacc 300 accgtgttca ccggccagtg cttcatcgaccgcaacggca aggaggtgct caagaccatg 360 tggctcctga ggagctccgt gaatgacatcggcgacgact ggaaggccac ccgcgtgggc 420 atcaacatct tcacccgcct ccgcacccagaaggagtga 459 2 552 DNA Zea mays 2 atgtcccccg agcgccgccc cgtcgagatccgcccggcca ccgccgccga catggccgcc 60 gtgtgcgaca tcgtgaacca ctacatcgagacctccaccg tgaacttccg caccgagccg 120 cagaccccgc aggagtggat cgacgacctggagcgcctcc aggaccgcta cccgtggctc 180 gtggccgagg tggagggcgt ggtggccggcatcgcctacg ccggcccgtg gaaggcccgc 240 aacgcctacg actggaccgt ggagtccaccgtgtacgtgt cccaccgcca ccagcgcctc 300 ggcctcggct ccaccctcta cacccacctcctcaagagca tggaggccca gggcttcaag 360 tccgtggtgg ccgtgatcgg cctcccgaacgacccgtccg tgcgcctcca cgaggccctc 420 ggctacaccg cccgcggcac cctccgcgccgccggctaca agcacggcgg ctggcacgac 480 gtcggcttct ggcagcgcga cttcgagctgccggccccgc cgcgcccggt gcgcccggtg 540 acgcagatct ga 552 3 309 DNA Zeamays 3 gccccgcagt ccatcaccga gctctgctcc gagtaccaca acacccagat ctacaccatc60 aacgacaaga tcctctccta caccgagagc atggccggca agcgcgagat ggtgatcatc 120accttcaagt ccggcgccac cttccaggtg gaggtgccgg gctcccagca catcgactcc 180cagaagaagg ccatcgagcg catgaaggac accctccgca tcacctacct caccgagacc 240aagatcgaca agctctgcgt gtggaacaac aagaccccga actccatcgc cgccatcagc 300atggagaac 309 4 382 DNA Hordeum vulgare 4 atggccaaca agcacctgagcctctccctc ttcctcgtgc tcctcggcct ctccgcctcc 60 ctcgccagcg gcgccccgcagtccatcacc gagctctgct ccgagtacca caacacccag 120 atctacacca tcaacgacaagatcctctcc tacaccgaga gcatggccgg caagcgcgag 180 atggtgatca tcaccttcaagtccggcgcc accttccagg tggaggtgcc gggctcccag 240 cacatcgact cccagaagaaggccatcgag cgcatgaagg acaccctccg catcacctac 300 ctcaccgaga ccaagatcgacaagctctgc gtgtggaaca acaagacccc gaactccatc 360 gccgccatca gcatggagaa ct382 5 1413 DNA Infectious pancreatic necrosis virus 5 aacaccaacaaggcaaccgc aacttacttg aaatccatca tgcttccaga gactggacca 60 gcaagcatcccggacgacat aacggagaga cacatcctaa aacaagagac ctcgtcatac 120 aacctagaggtctccgaatc aggaagtggc attcttgttt gtttccctgg ggcaccaggc 180 tcacggatcggtgcacacta cagatggaat gcgaaccaga cggggctgga gttcgaccag 240 tggctggagacgtcgcagga cctgaagaaa gccttcaact acgggaggct gatctcaagg 300 aaatacgacatccaaagctc cacactaccg gccggtctct atgctctgaa cgggacgctc 360 aacgctgccaccttcgaagg cagtctgtct gaggtggaga gcctgaccta caacagcctg 420 atgtccctaacaacgaaccc ccaggacaaa gtcaacaacc agctggtgac caaaggagtc 480 acagtcctgaatctaccaac agggttcgac aaaccatacg tccgcctaga ggacgagaca 540 ccccagggtctccagtcaat gaacggggcc aagatgaggt gcacagctgc aattgcaccg 600 cggaggtacgagatcgacct cccatcccaa cgcctacccc ccgttcctgc gacaggaacc 660 ctcaccactctctacgaggg aaacgccgac atcgtcaact ccacaacagt gacgggagac 720 ataaacttcagtctggcaga acaacccgca aacgagacca agttcgactt ccagctggac 780 ttcatgggccttgacaacga cgtcccagtt gtcacagtgg tcagctccgt gctggccaca 840 aatgacaactacagaggagt ctcagccaag atgacccagt ccatcccgac cgagaacatc 900 acaaagccgatcaccagggt caagctgtca tacaagatca accagcagac agcaatcggc 960 aacgtcgccaccctgggcac aatgggtcca gcatccgtct ccttctcatc agggaacgga 1020 aatgtccccggcgtgctcag accaatcaca ctggtggcct atgagaagat gacaccgctg 1080 tccatcctgaccgtagctgg agtgtccaac tacgagctga tcccaaaccc agaactcctc 1140 aagaacatggtgacacgcta tggcaagtac gaccccgaag gtctcaacta tgccaagatg 1200 atcctgtcccacagggaaga gctggacatc aggacagtgt ggaggacaga ggagtacaag 1260 gagaggaccagagtcttcaa cgaaatcacg gacttctcca gtgacctgcc cacgtcaaag 1320 gcatggggctggagagacat agtcagagga attcggaaag tcgcagctcc tgtactgtcc 1380 acgctgtttccaatggcagc accactcatc gga 1413 6 1485 DNA Hordeum vulgare 6 atggcgaacaagcacctgag ccttagcctc ttcctcgtgc tcctgggcct ctccgcctcc 60 ctcgcctccggcaacaccaa caaggcaacc gcaacttact tgaaatccat catgcttcca 120 gagactggaccagcaagcat cccggacgac ataacggaga gacacatcct aaaacaagag 180 acctcgtcatacaacctaga ggtctccgaa tcaggaagtg gcattcttgt ttgtttccct 240 ggggcaccaggctcacggat cggtgcacac tacagatgga atgcgaacca gacggggctg 300 gagttcgaccagtggctgga gacgtcgcag gacctgaaga aagccttcaa ctacgggagg 360 ctgatctcaaggaaatacga catccaaagc tccacactac cggccggtct ctatgctctg 420 aacgggacgctcaacgctgc caccttcgaa ggcagtctgt ctgaggtgga gagcctgacc 480 tacaacagcctgatgtccct aacaacgaac ccccaggaca aagtcaacaa ccagctggtg 540 accaaaggagtcacagtcct gaatctacca acagggttcg acaaaccata cgtccgccta 600 gaggacgagacaccccaggg tctccagtca atgaacgggg ccaagatgag gtgcacagct 660 gcaattgcaccgcggaggta cgagatcgac ctcccatccc aacgcctacc ccccgttcct 720 gcgacaggaaccctcaccac tctctacgag ggaaacgccg acatcgtcaa ctccacaaca 780 gtgacgggagacataaactt cagtctggca gaacaacccg caaacgagac caagttcgac 840 ttccagctggacttcatggg ccttgacaac gacgtcccag ttgtcacagt ggtcagctcc 900 gtgctggccacaaatgacaa ctacagagga gtctcagcca agatgaccca gtccatcccg 960 accgagaacatcacaaagcc gatcaccagg gtcaagctgt catacaagat caaccagcag 1020 acagcaatcggcaacgtcgc caccctgggc acaatgggtc cagcatccgt ctccttctca 1080 tcagggaacggaaatgtccc cggcgtgctc agaccaatca cactggtggc ctatgagaag 1140 atgacaccgctgtccatcct gaccgtagct ggagtgtcca actacgagct gatcccaaac 1200 ccagaactcctcaagaacat ggtgacacgc tatggcaagt acgaccccga aggtctcaac 1260 tatgccaagatgatcctgtc ccacagggaa gagctggaca tcaggacagt gtggaggaca 1320 gaggagtacaaggagaggac cagagtcttc aacgaaatca cggacttctc cagtgacctg 1380 cccacgtcaaaggcatgggg ctggagagac atagtcagag gaattcggaa agtcgcagct 1440 cctgtactgtccacgctgtt tccaatggca gcaccactca tcgga 1485 7 705 DNA Infectiouspancreatic necrosis virus 7 gacgaggagc tgcagcgcct cctgaacgcc acgatggccagggccaagga ggtccaggac 60 gccgagatct acaaacttct taagctcatg gcatggaccagaaagaacga cctcaccgac 120 cacatgtacg agtggtcaaa agaggacccc gatgcactaaagttcggaaa gctcatcagc 180 acgccaccaa agcaccctga gaagcccaaa ggaccagaccaacaccacgc ccaagaggcg 240 agagccaccc gcatatcatt ggacgccgtg agagccggggcggacttcgc cacaccggaa 300 tgggtcgcgc tgaacaacta ccgcggccca tctcccgggcagttcaagta ctacctgatc 360 actggacgag aaccagaacc aggcgacgag tacgaggactacataaaaca acccattgtg 420 aaaccgaccg acatgaacaa aatcagacgt ctagccaacagtgtgtacgg cctcccacac 480 caggaaccag caccagagga gttctacgat gcagttgcagctgtattcgc acagaacgga 540 ggcagaggtc ccgaccagga ccaaatgcaa gacctcagggagctcgcaag acagatgaaa 600 cgcaggccca ggaacgccga tgcgccacgc aggaccagggcgccagcgga accggcaccg 660 cccggacgct caaggttcac gcccagcgga gacaacgctgaggtg 705 8 777 DNA Hordeum vulgare 8 atggcgaaca agcacctgag ccttagcctcttcctcgtgc tcctgggcct ctccgcctcc 60 ctcgcctccg gcgacgagga gctgcagcgcctcctgaacg ccacgatggc cagggccaag 120 gaggtccagg acgccgagat ctacaaacttcttaagctca tggcatggac cagaaagaac 180 gacctcaccg accacatgta cgagtggtcaaaagaggacc ccgatgcact aaagttcgga 240 aagctcatca gcacgccacc aaagcaccctgagaagccca aaggaccaga ccaacaccac 300 gcccaagagg cgagagccac ccgcatatcattggacgccg tgagagccgg ggcggacttc 360 gccacaccgg aatgggtcgc gctgaacaactaccgcggcc catctcccgg gcagttcaag 420 tactacctga tcactggacg agaaccagaaccaggcgacg agtacgagga ctacataaaa 480 caacccattg tgaaaccgac cgacatgaacaaaatcagac gtctagccaa cagtgtgtac 540 ggcctcccac accaggaacc agcaccagaggagttctacg atgcagttgc agctgtattc 600 gcacagaacg gaggcagagg tcccgaccaggaccaaatgc aagacctcag ggagctcgca 660 agacagatga aacgcaggcc caggaacgccgatgcgccac gcaggaccag ggcgccagcg 720 gaaccggcac cgcccggacg ctcaaggttcacgcccagcg gagacaacgc tgaggtg 777

What is claimed is:
 1. A plant comprising a recombinant nucleotidesequence encoding an amino acid sequence which, when administered to afish, results in an antigenic or immunogenic response in said fish.
 2. Aseed of the plant of claim
 1. 3. Plant cells of the plant of claim
 1. 4.The plant of claim 1 wherein the amino acid sequence is an antigen of anorganism that causes disease or pathology in a fish.
 5. The plant ofclaim 1 wherein the nucleotide sequence is selected from the groupconsisting of SEQ ID NOS: 5, 6, 7, and
 8. 6. A composition foradministration to a fish, comprising plant material comprising anucleotide sequence encoding an amino acid sequence which, whenadministered to a fish, results in an antigenic or immunogenic responsein said fish.
 7. The composition of claim 6 wherein the plant materialcomprises seed tissue.
 8. The composition of claim 6 wherein the plantmaterial is combined with at least one nutrient or excipient.
 9. Thecomposition of claim 6 wherein the amino acid sequence is an antigen ofan organism that causes disease or pathology in fish.
 10. Thecomposition of claim 9 wherein the nucleotide sequence is selected fromthe group consisting of SEQ ID NOS: 5, 6, 7, and
 8. 11. A method ofproducing a composition for administration to a fish, comprisingtransforming a plant with a nucleotide sequence encoding an amino acidsequence which, when administered to a fish, results in an antigenic orimmunogenic response in said fish.
 12. The method of claim 11 whereinthe amino acid sequence an antigen of an organism that causes disease orpathology in fish.
 13. The method of claim 11 wherein the amino acidsequence is extracted from the plant.
 14. The method of claim 11 whereinthe transgenic plant is crossed with at least one plant to produceprogeny comprising the antigenic or immunogenic amino acid sequence. 15.The method of claim 11 wherein the nucleotide sequence is selected fromthe group consisting of SEQ ID NOS: 5, 6, 7, and
 8. 16. A method ofproducing a composition for administration to a fish comprisingproviding biomass from a plurality of plants, of which at least certainplants comprise a heterologous nucleotide sequence encoding an aminoacid sequence which, when administered to a fish, results in anantigenic or immunogenic response in said fish, wherein the nucleotidesequence is operably linked to a promoter to effect expression of theprotein by the certain plants.
 17. The method of claim 16 wherein theamino acid sequence is an antigen of an organism that produces diseaseor pathology in fish.
 18. The method of claim 17 wherein the nucleotidesequence is selected from the group consisting of SEQ ID NOS: 5, 6, 7,and
 8. 19. A method of inducing an immune response in fish comprisingfeeding fish a plant or plant material from a plant comprising anucleotide sequence encoding an amino acid sequence which, whenadministered to a fish, results in an antigenic or immunogenic responsein said fish.
 20. The method of claim 19 wherein the amino acid sequenceis from an antigen of an organism causing disease or pathology in fish.21. The method of claim 20 wherein the nucleotide sequence is selectedfrom the group consisting of SEQ ID NOS: 5, 6, 7, and
 8. 22. A method oftreating a fish infected with an organism that produces disease orpathology in fish comprising administering to the fish the compositionof claim 9 whereby said composition contains an antigen from saidorganism.
 23. The method of claim 22 wherein said composition contains anucleotide sequence selected from the group consisting of SEQ ID NOS: 5,6, 7, and 8.